Modulation of pleiotrophin signaling by receptor-type protein tyrosine phosphatase beta/zeta

ABSTRACT

The mechanism by which pleiotrophin binds to the receptor protein tyrosine phosphatase β/ζ(RPTP β/ζ) is disclosed along with methods of modulating both pleiotrophin expression and signaling to treat, prevent and inhibit abnormal cell growth states. Specifically provided are methods of inhibiting tumor growth, promotion, metastasis, invasiveness and angiogenesis as well as methods of preventing or inhibiting cell adhesion.

This application claims priority to copending U.S. provisional patentapplication Ser. No. 60/185,653, filed Feb. 29, 2000, incorporatedherein by reference.

BACKGROUND OF THE INVENTION

Pleiotrophin (PTN) is a platelet-derived growth factor-inducibleheparin-binding growth and differentiation factor that signals diversephenotypes in normal and deregulated cellular growth anddifferentiation. See Milner, et al., (1989) Biochem. Biophys. Res.Commun. 165, 1096–1103; Rauvala, H. (1989) EMBO J. 8(10), 2933–2941; Liet al. (1990) Science 250, 1690–1694; Li et al., (1992) Biochem.Biophys. Res. Commun. 184: 427–432. PTN is nearly 50% identical with theretinoic acid-inducible factor midkine, which is also a growth anddifferentiation factor active in cultured fibroblasts, endothelial cellsand epithelial cells. See Li et al., 1990, supra; Muramatsu etal.,(1993) Dev. Biol. 159, 392–402. Pleiotrophin gene expression islimited to specific cell types at different times during development;however, in adults, pleiotrophin gene expression is constitutive andlimited to only a few cell populations except in sites of injury, whenits expression is sharply increased. See Li et al. 1990, supra; Li etal., 1992 supra; Silos-Santiago et al, (1996) J. Neurobiol. 31, 283–296;Yeh et al., (1998) J. Neurosci. 18: 3699–3707.

The pleiotrophin (PTN) gene (Ptn) encodes an 18-kDa protein that ishighly conserved among mammalian species and that functions as a weakmitogen and promotes neurite-outgrowth activity in vitro. Chauhan etal., Proc. Nat'l. Acad. Sci. 90: 679–682, 1993. PTN cDNA encodes alysine-rich, highly basic protein of 168 amino acids with a 32-aminoacid signal sequence that is highly conserved in bovine, rat, human, andchicken. Zhang et al. J. Biol. Chem. 272:16733–16736, 1997. Thepleiotrophin gene is highly conserved among human, rat, bovine, andmouse species, and is developmentally regulated. Li et al., Biochem.Biophys. Res. Common. 184, 427–432, 1992. Li et al. (Science 250,1690–1694, 1990) reported the isolation and sequence of the frill-lengthcomplementary DNA's (cDNA's) of the bovine, human, and rat genes of aheparin binding protein (i.e., pleiotrophin) with mitogenic activitytoward rat and mouse fibroblasts. Comparison of predicted amino acidsequences of PTN from bovine, human, and rat revealed that PTN isconserved across the three species. Of 168 amino acid residues of PTN,163 between bovine and human and 164 between rat and human areidentical. The mature forms of bovine, human, and rat PTN exhibitoverall 98 percent sequence similarity. Li et al., Science 250,1690–1694, 1990. Zhang et al. describes the generation of a mouse PTNmutant gene construct containing sequences to encode mouse PTN residues−32 to +40 and a human wild type PTN expression vector containing afull-length human PTN cDNA fragment, and states that amino acid residues1–40 (after cleavage of the signal peptide) of mouse and human PTN areidentical, and thus the truncated PTN is equally effective in mouse andhuman lines. Zhang et al., J. Biol. Chem. 272: 16733–16736, 1997.

PTN also signals transformation; stable expression of an exogenous Ptngene transforms NIH 3T3 cells and the Ptn-transformed NIH 3T3 cells formrapidly growing highly vascularized tumors in nude mice. Chauhan et al.,(1993) PNAS USA 90: 679–682. Significantly, high level expression of thePtn gene is found in many different human malignant tumors and in thecell lines that have been derived from these tumors; however, Ptn geneexpression is not found in the normal cells from which the malignancy isderived. Fang, Hartmann et al. 1992, J. Biol. Chem. 267: 25889–97;Wellstein, Fang et al. 1992 J. Biol. Chem. 267: 2582–87; Tsutsui,Kadomatsu et al. 1993 Cancer Res. 53: 1281–85; Czubayko, Riegel et al.1994, J. Biol. Chem. 269: 21358–63; Czubayko, Schulte et al. 1995,Breast Cancer Res Treat 36: 157–68; Czubayko, Schulte et al. 1996 PNASUSA 93: 14753–58; Brodeur, Nakagawara et al. 1997 J. Neurooncol. 31:49–55; Zhang, Zhong et al. 1997 J. Biol. Chem. 272: 16733–36; Zhang andDeuel 1999 Curr Opin Hematol 6:44–50. Furthermore, high level expressionof the Ptn gene may play a important role in developing a moreaggressive phenotype in cancerous cells. Since it has also been shownthat interruption of endogenous PTN signaling by a dominant negative PTNeffector or a specific ribozyme reverses the malignant phenotype ofhuman breast cancer cells (Zhang et al. 1997, J. Biol. Chem. 16733–36)and human melanoma cells (Czukayko et al., 1994 J. Biol. Chem. 269:21358–63; Czubayko et al., 1996 PNAS USA 93: 14753–58), acquisition ofPTN signaling during the course of these malignancies may trigger a moreaggressive phenotype.

It is known that cells rely, to a great extent, on extracellularmolecules as a means by which to receive stimuli from their immediateenvironment. These extracellular signals are important in the regulationof diverse cellular processes such as differentiation, contractility,secretion, cell division, cell migration, contact inhibition andmetabolism. The extracellular molecules include, for example, hormones,growth factors or neurotransmitters, which may function as ligands thatbind specific cell surface receptors. The binding of these ligands totheir receptors triggers signal transduction, a cascade of reactionsthat brings about both the amplification of the original stimulus andthe coordinate regulation of the separate cellular processes mentionedabove.

A central feature of signal transduction is the reversiblephosphorylation of certain proteins. The phosphorylation ordephosphorylation of certain amino acid residues may triggerconformational changes in regulated proteins which results in thealteration of their biological properties. Proteins are phosphorylatedby protein kinases and are dephosphorylated by protein phosphatases.Phosphorylation is a dynamic process involving competing phosphorylationand dephosphorylation reactions, and the level of phosphorylation at anygiven instant reflects the relative activities, at that particularinstant, of the protein linases and phosphatases that catalyze thesereactions.

Protein kinases and phosphatases are classified according to the aminoacid residues they act on, for example, the class of tyrosine kinasesand phosphatases act on tyrosine residues. See Fischer, E. H. et al.,(1991) Science 253: 401–406; Schlessinger, J. and Ullrich, A., (1992)Neuron 9:383–391; Ullrich, A. and Schlessinger, J., (1990) Cell61:203–212. Protein kinases and phosphatases may further be defined asbeing receptors, i.e., the enzymes are an integral part of atransmembrane, ligand-binding molecule, or as non-receptors, meaningthey respond to an extracellular molecule indirectly by being acted uponby a ligand-bound receptor.

The receptor class of protein tyrosine phosphatases (PTPs) is made up ofhigh molecular weight, receptor-linked PTPases, termed RPTPases.Structurally resembling growth factor receptors, RPTPases consist of anextracellular, putative ligand-binding domain, a single transmembranesegment, and an intracellular catalytic domain (reviewed in Fischer etal., (1991) Science 253:401–406). Since the initial purification,sequencing and cloning of a protein tyrosine phosphatase (Thomas, M. L.et al., (1985) Cell 41:83), additional potential protein tyrosinephosphatases have been identified. One such example is aproteoglycan-type protein tyrosine phosphatase, named protein tyrosinephosphatase ζ/receptor-like PTP β (RPTP β/ζ). Recently, PTN was found tointeract with the transmembrane RPTP β/ζ. See Maeda et al., (1996) J.Biol. Chem. 271: 21446–21452: Maeda, N. & Noda, M. (1998) J. Cell Biol.142, 203–216; Milev et al., (1998) J. Biol. Chem. 273: 6998–7005.

The PTN gene is a protooncogene and is expressed in many human tumorssuch as breast cancer, neuroblastoma, glioblastoma, prostate cancer,lung cancer and Wilms' tumor and cell lines derived from human tumors.See Fang et al., (1992) J. Biol. Chem. 267: 25889–25897; Chauhan et al,(1993) Proc. Natl. Acad. Sci. USA 90: 679–682; Wellstein et al., (1992)J. Biol. Chem. 267: 2582–2587; Tsutsui et al., (1993) Cancer Res.53:1281–1285; Nakagawara et al., (1995) Cancer Res. 55: 1792–1797. Theimportance of PTN in malignant cell growth was first established whenintroduction of the exogenous Ptn gene into NIH 3T3 cells and NRK cellsled to morphological transformation, anchorage independent growth andtumor formation with significant neovascularization in vivo in the nudemouse. See Chauhan et al., 1993 PNAS USA 90: 679–82. It was subsequentlyshown that SW13 cells transformed by pleiotrophin also develop highlyvascular tumors in the flanks of athymic nude mice. See Fang et al.,1992 J. Biol. Chem. 267: 258889–97. Further, interruption of PTNsignaling has resulted in the reversal of the transformed phenotype ofhuman breast cancer cells that constitutively express the PTN gene(Zhang et al., (1997) J. Biol. Chem. 272: 16733–16736) and effectivelyreverted the malignant phenotype of cultured human melanoma cells(Czubayko et al, (1994) J. Biol. Chem. 269: 21358–21363). It is believedthat expression of the Ptn gene and its signaling pathway play a crucialregulatory role in many neoplasms of diverse origins. Thus,identification of the molecules and mechanisms of the PTN signalingpathway that are specific and crucial for tumor proliferation,angiogenesis and invasiveness would allow for the development ofclinical applications and specific anti-tumor drugs to treat cancer. Assuch, a need presently exists for the identification of compounds oragents that disrupt or interfere with PTN signaling in order toinfluence malignant transformation and inhibit tumor growth andangiogenesis.

Further, PTN also induces neurite outgrowth from neurons (Rauvala 1989,supra; Li et al. 1990, supra) and glial process outgrowth from glialprogenitor cells, suggesting that Ptn gene expression may influence avery broad range of functional activities. Since the pleiotrophin geneexpression is upregulated by PDGF, PTN may act downstream of PDGF tomediate aspects of the PDGF signal. Thus, the activation of theirrespective signaling pathways is critical to the temporal maturation ofoligodendrocyte progenitors and the properties of PTN suggest that PTNis ideally positioned to signal activation of genes important inmaturation of glial elements at this critical time of development. Asdifferentiation of oligodendrocytes is required for myelination of nervefibers and consequently, important to nerve conduction, thedetermination of mechanisms for modulating PTN signaling during thedifferentiation of oligodendrocytes would be desirable. Accordingly, aneed presently exists to determine the mechanism and molecules by whichPTN signals in order to develop methods to treat and prevent nerveinjury and demyelinating diseases.

While the molecules through which PTN signals have not to date beenestablished, in addition to interacting with RPTP β/ζ, PTN has also beenshown to bind to heparin, heparin sulfate proteoglycans andextracellular matrix. See Milner et al. 1989, supra; Rauvala, 1989supra; Li et al., 1990, supra; Raulo et al., (1994) J. Biol. Chem. 269:12999–13004; Maeda et al., (1996) J. Biol. Chem. 271: 21446–21452;Kinnunen et al., (1996) J. Biol. Chem. 271: 2243–2248. In addition tointeracting with RPTP β/ζ, PTN induces tyrosine phosphorylation of a 190kDa protein in PTN treated murine fibroblasts. See Li, Y. S. & Deuel, T.F. (1993) Biochem. Biophys. Res. Commun. 195: 1089–1095.

Thus, the interruption of PTN signaling impacts the events downstream inthe signaling cascade such as cell proliferation and differentiation.Accordingly, there is presently a need to understand PTN signaling andthe interaction between RPTP β/ζ and PTN in order to modulate the PTNsignaling pathway to produce increased or decreased PTN activity inorder to define compounds which useful in therapy and treating diseaseinfluenced by the expression of pleiotrophin such as cancer.

SUMMARY OF THE INVENTION

Applicants have shown that receptor protein tyrosine phosphatase β/ζ(RPTP β/ζ) is the receptor for pleiotrophin (PTN). Binding of RPTP β/ζand PTN inhibits RPTP β/ζ enzymatic activity and results in higherlevels of tyrosine phosphorylation of β-catenin. Further, binding ofRPTP β/ζ and PTN also reduces the levels of the β-catenin interactionwith E-cadherin and thus affects the potential for cells to adhere witheach other.

The elucidation of this relationship between RPTP β/ζ and PTN can beused to define compounds which useful in therapy and treating disease.For example, this pathway can be modulated to mimic increased PTNactivity in order to promote glial process formation, neuron growth anddifferentiation, endothelial cell growth and differentiation, andfibroblast growth. The method of accomplishing these effects involvesthe use of agents which either (a) mimic PTN binding to RPTP β/ζ, (b)inhibit the binding of RPTP β/ζ to B-catenin, (c) enhance or increasethe binding or the amounts of phosphorylated β-catenin to LEF-1 to forma transcription factor, or (d) mimic PTN binding to RPTP β/ζ.

Thus, among the several aspect of the present invention, therefore,include methods of monitoring levels of tyrosine phosphatase activity ofRPTP β/ζ in a cell or tissue comprising contacting the cell or tissuewith an effective amount of pleiotrophin which binds to the active siteof RPTP β/ζ thereby reducing tyrosine phosphatase activity of RPTP β/ζ.Preferably, the administration of pleiotrophin results in the increaseof PTN activity and the reduction of tyrosine phosphatase activity ofRPTP β/ζ. Furthermore, the binding of pleiotrophin to the active site ofRPTP β/ζ preferably results in ligand-dependent dimerization of RPTP β/ζand inactivates the catalytic activity of RPTP β/ζ.

Another aspect of the present invention is directed to methods ofregulating levels of tyrosine phosphatase activity of protein tyrosinephosphataseζ/receptor-like protein tyrosine phosphatase β (RPTP β/ζ) ina cell or tissue, the method comprising:

-   -   a. determining whether the tyrosine phosphatase activity should        be reduced or increased in the cell or tissue to effectuate a        desired physiologic change;    -   b. administering an effective amount of pleiotrophin,        pleiotrophin inhibitor or mimic to reduce or increase the        tyrosine phosphatase activity of RPTP β/ζ;    -   c. monitoring the cell or tissue for the appearance of the        desired physiologic change; and    -   d. determining whether to further modify levels of tyrosine        phosphatase activity.

Yet another aspect of the present invention is directed to methods ofincreasing tyrosine phosphorylation of β-catenin in a cell or tissuecomprising contacting the cell or tissue that expresses RPTP β/ζ with aneffective amount of pleiotrophin thereby reducing tyrosine phosphataseactivity of RPTP β/ζ and increasing tyrosine phosphorylation ofβ-catenin.

In another aspect, methods for modulating cell-cell adhesion areprovided which include contacting a cell with pleiotrophin in an amountsufficient to inactivate tyrosine phosphatase activity of RPTP β/ζthereby increasing tyrosine phosphorylation of β-catenin in the cell anddecreasing interaction of β-catenin and E-cadherin.

The PTN signaling pathway can also be modulated to mimic reduced PTNactivity to prevent or inhibit the growth or promotion of tumor cellsand the loss of cell-cell interactions in cancer. This could beaccomplished by agents that (a) reduce or block PTN binding to RPTP β/ζ,(b) ensure the binding of RPTP β/ζ to β-catenin and its ability tomaintain normal steady state levels of tyrosine phophorylation ofβ-catenin, (c) reduce or eliminate the binding of phosphorylatedβ-catenin to LEF-1 to form a transcription factor, or (d) reduce oreliminate the translocation of phosphorylated β-catenin to the nucleus.Agents with these activities can be identified by screening chemicallibraries in in vitro assays as described in the Examples herein.

Thus, another aspect of the present invention is directed to methods ofinhibiting tumor invasiveness in a tissue comprising contacting thetissue with an effective amount of a compound which binds to RPTP β/ζ orpleiotrophin thereby preventing pleiotrophin from binding to RPTP β/ζand decreasing tyrosine phosphatase activity of RPTP β/ζ.

Relatedly, the invention is further directed to methods of inhibitingmetastasis of a tumor comprising contacting the tumor with an effectiveamount of a compound which binds to pleiotrophin or RPTP β/ζ in thetissue thereby preventing pleiotrophin from binding to RPTP β/ζ andincreasing tyrosine phosphatase activity of RPTP β/ζ.

Yet another method of inhibiting tumor angiogenesis, progression orpromotion includes reducing the level of PTN signaling through RPTP β/ζin the tumor cells.

Further, methods of inhibiting tumor growth in a mammal includeadministering to the mammal an effective amount of a compound whichbinds to pleiotrophin or RPTP β/ζ thereby reducing the level ofpleiotrophin signaling through RPTP β/ζ in the tumor cells.

Also provided are pleiotrophin mimics which are compounds which bind topleiotrophin or RPTP β/ζ in manner which corresponds to the effectivebinding of PTN to RPTP β/ζ in a cell or tissue.

Other aspects and features will be in part apparent and in part pointedout hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B and 1C are a set of three western blots showing theassociation of RPTP β/ζ with PTN. FIG. 1A shows lysates of U373-MGglioblastoma cells immunoprecipitated with anti-RPTP β/ζ monoclonalantibodies. The immunoprecipitates were separated on 6% acrylamide gel,transferred to a poly(vinylidene difluoride) membrane, and probed withanti-RPTP β/ζ antibodies. The arrowheads indicate the RPTP β/ζ-splicedproducts of ≈230, 130 and 85 kDa. FIG. 1B shows Western analysis of RPTPβ/ζ captured by PTN-Fc. Lysates of U373-MG cells were incubated withPTN-Fc and proteins interactive with PTN-Fc (right lane) were capturedwith Protein A Sepharose-4B beads for 2 hours. The beads were washed incold lysis buffer, boiled in SDS/PAGE sample buffer, and the elutedproteins were separated on an 8% acrylamide gel and analyzed by Westernblots probed with anti-RPTP β/ζ monoclonal antibodies. As a control,PTN-Fc was replaced with an equal amount of human IgG (left lane). Thearrowheads indicate the ≈130 and ≈85 kDa-spliced products of RPTP β/ζ.FIG. 1C shows western analysis of RPTP β/ζ captured by endogenous PTN.Lysates of U373-MG cells were incubated with anti-PTN monoclonalantibodies (right lane) and the complexes were captured with Protein ASepharose-4B beads for 2 hours. The beads were washed in cold lysisbuffers, boiled in SDS-PAGE sample buffer, and the eluted proteins wereseparated on an 8% acrylamide gel and analyzed by Wester blots probeswith anti-RPTP β/ζ monoclonal antibodies. As a control, mouse IgGreplaced the anti-PTN antibody (left lane). The arrowheads indicate the130 and =85 kDa-spliced products of RPTP β/ζ.

FIGS. 2A, 2B and 2C are a set of three bar charts showing PTN-dependentinhibition of the intrinsic tyrosine phosphatase activity of RPTP β/ζ.FIG. 2A shows inhibition of the endogenous RPTP β/ζ tyrosine phosphataseactivity in PTN-treated U373-MG cells. The left bar represents tyrosinephosphatase activity in immunoprecipitates from lysates of untreatedcells with mouse IgG (control) to replace the anti-RPTP β/ζ antibodies.The center bar represents tyrosine phosphatase activity inimmunoprecipitates with anti-RPTP β/ζ antibodies from lysates ofuntreated cells, and the right bar represents tyrosine phosphataseactivity of immunoprecipitates with anti-RPTP β/ζ antibodies fromlysates of cells treated with recombinant PTN (50 ng/ml). FIG. 2B showsinhibition of recombinant RPTP β/ζ phasphatase activity in Sf9 cellmembranes. The right two bars show membrane fractions of Sf9 cells thatwere infected by a baculovirus containing a cDNA-encoding RPTP β/ζ, orwere uninfected (left two bars) that were untreated (−PTN) or treated(+PTN) with 50 ng/ml PTN. FIG. 2C shows a time course of PTN-dependentinactivation of RPTP β/ζ in PTN-treated (50 ng/ml) Sf9 cell membranesexpressing RPTP β/ζ (solid bars) and SF9 cell membranes without RPTP β/ζ(open bar, t=0 only).

FIGS. 3A and 3B are a set of two (FIG. 3A) and one (FIG. 3B) Westernblots, respectively, showing physical and functional association ofβ-catenin with PTN/RPTP β/ζ. FIG. 3A shows that PTN-Fc is in complexwith RPTP β/ζ and β-catenin. PTN-Fc treated confluent U373-MG cells from60-mm dish were chemically cross-linked with 3,3′dithiobissulfosuccinmidyl propionate. Lysates from PTN-Fc-treated, chemicallycross-linked cells (lanes 1) or Fc-(alone) treated (control) U373-MGcells (lane 2) were incubated with Protein A Sepharose, washed, elutedwith SDS sample buffer with 5% 2-mercaptoethanol, and analyzed in 6% SDSgels and Western blots. Lysates from untreated U373-MG cells alone (lane3) were also analyzed as a control. Western blots were analyzed withanti-β-catenin (right) or anti-RPTP β/ζ antibodies (left). Arrowheadsidentify RPTP β/ζ-spliced products of =250, 230, 180 and 85 kDa (left)and β-catenin (94 kDa) (right). FIG. 2B shows that β-catenin interactswith proximal (catalytic) domain of RPTP β/ζ. The GST-D1-RPTP β/ζwild-type, GST-D1-Cys-1925-Ser (inactivating) mutant fusion protein orGST alone were expressed and immobilized with glutathione-Sepharose-48beads, incubated with U373-MG cell lysates, washed, and analyzed inWestern analysis with the a-phosphotyrosine antibodies and visualizedwith the enhanced chemiluminescence ECL-PLUS system (lower). The sameblot was reprobed with α-β-catenin antibodies and detected as above(upper).

FIGS. 4A and 4B are a pair of western blots showing increased β-catenintyrosine phosphorylation. FIG. 4A is a time course of the tyrosinephosphorylation of β-catenin in response to PTN-FC treatment. Cells weretreated with 10 ng/ml PTN-Fc for the times indicated. Lysates wereimmunoprecipitated with α-β-catenin antibodies and analyzed in Westernblots probed with α-phosphotyrosine anti9bodies (upper) and the blotswere reprobed with α-β-catenin antibodies and (lower). In FIG. 4B,U373-MG cells were treated with different doses of PTN-Fc for 20minutes. Cells were grown to near confluence, and then wereserum-starved for 48 hours. PTN-FC was added up to the indicatedconcentrations. The Fc fragment alone (20 ng/ml) was added as a control.Lysates were immunoprecipitated and analyzed in Western blots withantiphosphotyrosine antibodies as described above. Parallel immunoblotswere probed for β-catenin.

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description is provided to aid those skilled inthe art in practicing the present invention. Even so, this detaileddescription should not be construed to unduly limit the presentinvention as modifications and variations in the embodiments discussedherein can be made by those of ordinary skill in the art withoutdeparting from the spirit or scope of the present inventive discovery.

All publications, patents, patent applications, databases and otherreferences cited in this application are herein incorporated byreference in their entirety as if each individual publication, patent,patent application, database or other reference were specifically andindividually indicated to be incorporated by reference.

Abbreviations and Definitions

As used herein, “Ptn” refers to the pleiotrophin gene.

As used herein, PTN refers to the pleiotrohin protein.

The phrase “preventing or inhibiting interaction between RPTP β/ζ andPTN” indicates that the normal interaction between a RPTP β/ζ and PTN isbeing affected either by being inhibited or reduced to such an extentthat the binding of PTN to RPTP β/ζ is measurably lower than is the casewhen PTN is interacting with RPTP β/ζ at conditions which aresubstantially identical (with regard to pH, concentration of ions, andother molecules) to the native conditions in the cell or tissue.

By the phrase “effective amount” is meant the amount of a desiredcompound necessary to (a) inhibit PTN binding to RPTP β/ζ and inhibitsits intrinsic catalytic activity, (b) inhibit the binding of RPTP β/ζ toβ-catenin, (c) inhibit the binding of phosphorylated β-catenin to LEF-1to form a transcription factor, or (d) mimic PTN binding to RPTP β/ζ.

By the term “a mimic of PTN” denotes any substance which mimics or hasthe ability to bind to pleiotrophin or to RPTP β/ζ in a manner whichprevents the effective binding of PTN to RPTP β/ζ in a cell or tissue.Such a mimic of PTN can be a modified form of the intact PTN or it canbe a modified form of the protein which may be coupled to a probe,marker or another moiety. Another such mimic can be obtained bymodifying or mutating PTN so that it differs from the wild-type sequenceencoding PTN by the substitution of at least one amino acid residue ofthe wild-type sequence with a different amino acid residue and/or by theaddition and/or deletion of one or more amino acid residues to or fromthe wild-type sequence. The additions and/or deletions can be from aninternal region of the wild-type sequence and/or at either or both ofthe N- or C-termini. In the present context, PTN and mimics thereofexhibit at least one binding characteristic relevant for the interactionof PTN and RPTP β/ζ during PTN signaling in a cell or tissue. Suchmimics and compounds can also be small molecules which have the effectsof the mimicking factor described above.

The term “ligand-dependent receptor inactivation of RPTP β/ζ” refers tothe mechanism in PTN signaling pathways by which PTN binds to RPTP β/ζ,inactivates the catalytic tyrosine phosphatase activity of RPTP β/ζ anddisrupts the normal role of RPTP β/ζ in the regulation of steady-statetyrosine phosphorylation of downstream signaling molecules.

In accordance with the present invention, applicant has identified thatPTN is a natural ligand for RPTP β/ζ. PTN is the first natural ligandidentified for any of the RPTP family and its identification provides aunique tool to pursue both the novel signaling pathway activated by PTNand the relationship of PTN signaling with other pathways regulatingβ-catenin. Furthermore, the finding of RPTP β/ζ as the functionalreceptor for PTN is particularly interesting because to date, there areno known soluble ligands for this class of transmembrane receptortyrosine phosphatases and thus, PTN may be a unique probe for exploringthe receptor class of transmembrane tyrosine phosphatases and how theysignal.

Without intending to be bound by any particular theory, it is believedthat PTN signals through “ligand-dependent receptor inactivation” ofRPTP β/ζ and disrupts its normal roles in the regulation of steady-statetyrosine phosphorylation of downstream signaling molecules.Specifically, PTN binds to RPTP β/ζ, inducing ligand-dependentdimerization of RPTP β/ζ and functionally inactivates the catalytictyrosine phosphatase activity of RPTP β/ζ, presumably denying the accessof substrate(s) to its catalytic site. An active site-containing domainof RPTP β/ζ both binds β-catenin and functionally reduces its levels oftyrosine phosphorylation when added to lysates of pervanidate-treatedcells. Thus, this mechanism of PTN signaling through RPTP β/ζ providesfurther insight into the mechanism by which PTN affect downstreamsignaling.

Further, β-catenin interacts with the catalytically active D1 domain ofRPTP β/ζ and addition of the D1 domain of RPTP β/ζ with an activetyrosine phosphatase catalytic site to lysates of cells previouslytreated with pervanidate sharply reduces levels of tyrosinephosphorylation of β-catenin; it is believed that β-catenin is asubstrate for the tyrosine phosphatase activity of RPTP β/ζ.Furthermore, because PTN rapidly signals tyrosine phosphorylation ofβ-catenin in intact U373-MG cells, inactivation of RPTP β/ζ is believedto be directly responsible for the increase in tyrosine phosphorylationof β-catenin as a result of the disruption of the normal balance oftyrosine kinase and phosphatase activities. Thus, RPTP β/ζ isintrinsically active and a principal regulator of tyrosinephosphorylation levels of β-catenin. In PTN-stimulated cells, RPTP β/ζis believed to be functionally inactivated, steady-state levels ofβ-catenin tyrosine phosphorylation and other downstream signalingmolecules are increased and a PTN-dependent downstream signaling cascadeis initiated. Thus, β-catenin not only is an endogenous substrate forRPTP β/ζ, but also a downstream mediator of PTN signaling.

Accordingly, the elucidation of this relationship between RPTP β/ζ andPTN can be used to define compounds which useful in therapy and treatingdisease. For example, this pathway can be modulated to mimic increasedPTN activity in order to promote glial process formation, neuron growthand differentiation, endothelial cell growth and differentiation, andfibroblast growth. The method of accomplishing these effects involvesthe use of agents which either (a) mimic PTN binding to RPTP β/ζ, (b)inhibit the binding of RPTP β/ζ to β-catenin, (c) enhance or increasethe binding or the amounts of phosphorylated β-catenin to LEF-1 to forma transcription factor, or (d) mimic PTN binding to RPTP β/ζ.

Thus, one aspect of the present invention provides methods of regulatingand/or modifying levels of tyrosine phosphatase activity of RPTP β/ζ ina cell or a tissue. Such methods include determining whether thetyrosine phosphatase activity should be reduced or increased in the cellor tissue to effectuate a desired physiologic change; administering aneffective amount of pleiotrophin, pleiotrophin inhibitor or pleiotrophinmimic to reduce or increase the tyrosine phosphatase activity of RPTPβ/ζ; monitoring the cell or tissue for the appearance of the desiredphysiologic change; and determining whether to further modify levels oftyrosine phosphatase activity. Such desired physiological changesinclude but are not limited to tumor promotion, growth angiogenesis,metastasis, modulation of cell-cell adhesion and differentiation ofoligodendrocytes.

In another embodiment, the present invention provides methods formonitoring tyrosine phosphatase activity of RPTP β/ζ in a cell ortissue. This method involves contacting the cell or tissue with aneffective amount of pleiotrophin which binds to RPTP β/ζ, preferably theactive site of RPTP β/ζ. Preferably, in cells which express PTN,administering pleiotrophin results in the reduction of tyrosinephosphatase activity of RPTP β/ζ. Furthermore, the binding ofpleiotrophin to the active site of RPTP β/ζ preferably results inligand-dependent dimerization of RPTP β/ζ and inactivates the catalyticactivity of RPTP β/ζ.

Further, an (inactivating) active-site mutant of RPTP β/ζ also bindsβ-catenin but fails to reduce tyrosine phosphorylation of β-catenin. Inparallel to its ability to inactivate endogenous RPTP β/ζ, PTN increasestyrosine phosphorylation of β-catenin in PTN-treated cells. Thus, inunstimulated cells, RPTP β/ζ is intrinsically active and functions as animportant regulator in the reciprocal control of the steady statetyrosine phosphorylation levels of β-catenin by tyrosine kinases andphosphatases. As such, it is believed that RPTP β/ζ is a functionalreceptor for PTN and that PTN signals through ligand-dependent receptorinactivation of RPTP β/ζ to increase levels of tyrosine phosphorylationof β-catenin to initiate downstream signaling.

Formation of cell-cell adhesion requires members of the cadherin-cateninfamilies to link the highly conserved cadherin cytoplasmic domain to theactin-based cytoskeleton and to connect adjacent cells via the cadherinextracellular domains. See Kypta et al., (1996) J. Cell Biol. 134:1519–1529; Tonks, N. K. & Neel, B. G. (1996) Cell 87: 365–368; Miller,J. R., & Moon, R. T. (1996) Genes Dev. 10, 2527–2537. Balsamo et al. (J.Cell. Biol. 134: 801–813 (1996)) demonstrated that the association ofβ-catenin with E-cadherin is inversely related to tyrosinephosphorylation levels of β-catenin in pervanidate-treated cells,raising the distinct possibility that through its ability to increasetyrosine phosphorylation of β-catenin, PTN disrupts the normalassociation of β-catenin and E-cadherin, underscoring the need forreciprocal control of tyrosine phosphorylation of β-catenin. Kypta etal.,(1996) J. Cell Biol. 134: 1519–1529; Hoschuetzky et al.,(1994) J.Cell Biol. 127:1375–1380; Fischer et al.,(1991) Science 253: 401–406;Brady-Kalnay et al.,(1995) J. Cell. Biol. 130: 977–986; Brady-Kalnay etal., (1998) J. Cell. Biol. 141: 287–296. Because constitutive expressionof PTN itself transforms cells with striking loss of contact inhibition,cell adhesion, and striking disruption of cytoskeletal architecture, itis believed that the ability of PTN to disrupt the reciprocal control oftyrosine phosphorylation of β-catenin by tyrosine kinases andphosphatases may account for many of the properties of PTN-transformedcells and those human cancer cells which constitutively express PTN.

Thus, in a preferred embodiment of the invention, tyrosinephosphorlyation of β-cateninin a cell or tissue is increased, preferablyin tissues or cells that express PTN. Preferably, increasing levels oftyrosine phosphorlyation of β-catenin in a cell or tissue reduces thelevel of β-catenin interaction with E-cadherin thus affecting cell-celladhesion. Loss of cell-cell interactions in cancer have a profoundeffect on tumor formation, promotion, angiogenesis and metastatsis.Preferably, increasing the levels of tyrosine phosphorlyation ofβ-catenin in a cell or tissue reduces the level of β-catenin interactionwith E-cadherin and more preferably, affects the potential for cells toadhere with each other. Hence, in a preferred embodiment, methods ofincreasing the levels of tyrosine phosphorlyation of β-catenin in a cellor tissue by administering pleiotrophin will affect cell-cell adhesion,preferably, by increasing the levels of tyrosine phosphorlyation ofβ-catenin to prevent or inhibit cell-cell adhesion. These methodsinvolve contacting the cell or tissue with an effective amount ofpleiotrophin thereby reducing tyrosine phosphatase activity of RPTP β/ζand increasing tyrosine phosphorylation of β-catenin. Preferably,pleiotrophin inactivates tyrosine phosphatase activity of RPTP β/ζ bybinding to the active site of RPTP β/ζ and more preferably, inducesligand-dependent dimerization of RPTP β/ζ. In a preferred embodiment,the ligand-dependent dimerization of RPTP β/ζ inhibits the ability ofβ-catenin to bind to the catalytic site of RPTP β/ζ, preferably the D1site of RPTP β/ζ.

In another aspect of the invention, the PTN signaling pathway can bemodulated to mimic reduced PTN activity thereby impacting eventsdownstream in the signaling cascade such as inhibiting the growth,proliferation, promotion, angiogenesis and/or metastatsis of tumor cellsand the loss of cell-cell interactions in cancer. This can beaccomplished by agents that (a) reduce or block PTN binding to RPTP β/ζ,(b) ensure the binding of RPTP β/ζ to β-catenin and its ability tomaintain normal steady state levels of tyrosine phophorylation ofβ-catenin, (c) reduce or eliminate the binding of phosphorylatedβ-catenin to LEF-1 to form a transcription factor, or (d) reduce oreliminate the translocation of phosphorylated β-catenin to the nucleus.Further, it is believed that PTN also signals nuclear translocation andtransactivation of genes signaling oncogenic pathways as a consequenceof the release of β-catenin from E-cadherin. Desirable results ofdecreasing PTN signaling is the reduction or inhibition of tumor growth,promotion, proliferation, angiogenesis and metastasis. During the studyof PTN signaling, Applicant observed that PTN not only transformed NIH3T3 cells but that NIH-PTN cells established rapidly growing highlyvascularized tumors in nude mice, suggesting that PTN may promote tumorgrowth by inducing tumor angiogenesis. Subsequently, it was shown thatintroduction of PTN into human adrenal carcinoma cells increased thenumber of new blood vessels when these cells were implanted into theflanks of nude mice. Furthermore, it was also possible to show that thestimulation of angiogenesis in SW13 cells by constitutive expression ofPtn could be localized to a domain of PTN within PTN amino acid residues69–136.

Accordingly in a preferred embodiment of the invention, methods ofinhibiting tumor invasiveness in a tissue the method include contactingthe tissue with an effective amount of a compound which binds to RPTPβ/ζ or pleiotrophin thereby preventing pleiotrophin from binding to RPTPβ/ζ and decreasing tyrosine phosphatase activity of RPTP β/ζ.

In another embodiment, methods are provided which reduce the level ofPTN signaling through RPTP β/ζ in the tumor cells thus resulting in theinhibition of tumor angiogenesis, progression or promotion.

Yet another embodiment provides a method of inhibiting tumor growth in amammal comprising administering to the mammal an effective amount of acompound which binds to pleiotrophin or RPTP β/ζ thereby reducing thelevel of pleiotrophin signaling through RPTP β/ζ in the tumor cells.Preferably, the tumor cells are tumor cells from breast cancer,neuroblastoma, glioblastoma, prostate cancer, lung cancer and Wilms'tumor.

In the above methods, an effective amount of a compound which binds toRPTP β/ζ or pleiotrophin can be antibodies to RPTP β/ζ, antibodies topleiotrophin or a pleiotrophin mimic. Accordingly, in one aspect, thepresent invention directed to a compound, preferably a pleiotrophinmimic, which will mimic the capability of pleiotrophin to bind to RPTPβ/ζ, thereby modulating, disrupting or interfering with PTN signaling.The compound can be any compound, preferably a peptide, which will bindto pleiotrophin or RPTP β/ζ and prevent the binding of pleiotrophin toRPTP β/ζ. In a preferred embodiment, the pleiotrophin mimic is a peptidecompound. It will be appreciated that by virtue of the presentinvention, the polypeptide pleiotrophin mimic can be synthesized usingconventional synthesis procedures commonly used by one skilled in theart. For example, the polypeptides can be chemically synthesized usingan automated peptide synthesizer (such as one manufactured by PharmaciaLKB Biotechnology Co., LKB Biolynk 4170 or Milligen, Model 9050(Milligen, Millford, Mass.)) following the method of Sheppard, et al.,Journal of Chemical Society Perkin I, p. 538 (1981). In this procedure,N,N′-dicyclohexylcarbodiimide is added to amino acids whose aminefunctional groups are protected by 9-flourenylmethoxycarbonyl (Fmoc)groups and anhydrides of the desired amino acids are produced. TheseFmoc-amino acid anhydrides can then be used for peptide synthesis. AFmoc-amino acid anhydride corresponding to the C-terminal amino acidresidue is fixed to Ultrosyn A resin through the carboxyl group usingdimethylaminopyridine as a catalyst. Next, the resin is washed withdimethylformamide containing piperidine, and the protecting group of theamino functional group of the C-terminal acid is removed. The next aminoacid corresponding to the desired peptide is coupled to the C-terminalamino acid. The deprotecting process is then repeated. Successivedesired amino acids are fixed in the same manner until the peptide chainof the desired sequence is formed. The protective groups other than theacetoamidomethyl are then removed and the peptide is released withsolvent.

Alternatively, the polypeptides can be synthesized by using nucleic acidmolecules which encode the peptides of this invention in an appropriateexpression vector which include the encoding nucleotide sequences. SuchDNA molecules may be readily prepared using an automated DNA sequencerand the well-known codon-amino acid relationship of the genetic code.Such a DNA molecule also may be obtained as genomic DNA or as cDNA usingoligonucleotide probes and conventional hybridization methodologies.Such DNA molecules may be incorporated into expression vectors,including plasmids, which are adapted for the expression of the DNA andproduction of the polypeptide in a suitable host such as bacterium,e.g., Escherichia coli, yeast cell or mammalian cell.

It is known that certain modifications can be made without completelyabolishing the polypeptide's ability to bind to pleiotrophin or RPTPβ/ζ. Modifications include the removal and addition of amino acids.Polypeptides containing other modifications can be synthesized by oneskilled in the art and compounds comprising such polypeptides may betested for biological activity in the various assays and methodsdescribed in a later section. Thus, the effectiveness of thepolypeptides can be modulated through various changes in the amino acidsequence or structure.

Further, it should be understood that the mimic may be modified usingmethods known in the art to improve binding, specificity, solubility,safety, or efficacy. A necessary characteristic of these preferredcompounds is the capability to interact with pleiotrophin or RPTP β/ζ insuch a manner that PTN signaling is disrupted or interfering preventedor inhibited.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof the invention. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments that are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention, therefore all matter set forth or shown in the accompanyingdrawings is to be interpreted as illustrative and not in a limitingsense.

EXAMPLES Example 1 Materials and Methods

Cell Culture

U373-MG glioblastoma (American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va., 20110-2209, USA) cells were used in allexperiments and cultured in DMEM and 10% FCS unless otherwise noted.

Western Blot Analysis

U373-MG glioblastoma cells (≈10⁶) were lysed in 50 mM Tris-HCl (pH8.0)/150 mM NaCl/1 mM EDTA/1% Triton X-100/1 mM phenylmethylsulfonylfluoride/0.5 μg/ml leupeptin/1 μM pepstatin/1 μg/ml aprotinin for 30minutes at 4° C., boiled in SDS/PAGE sample buffer (25 mM Tris-HCl, pH6.8/2.5% SDS/2.5% glycerol/5% 2-mercaptoethanol), separated by SDS/PAGE,transferred to poly(vinylidenedifluoride) membranes, probed withantibodies as indicated, and illuminated with the enhancedchemiluminescence ECL-PLUS system (Amersham Corp., Arlington Heights,Ill., USA).

Chemical Cross-Linking

U373-MG cells (≈10⁶) were incubated with the PTN-Fc fragment of IgG(PTN-Fc) for 30 minutes at 37° C., washed with PBS, and incubated with 1mM of the reversible cross-linking agent 3,3′-dithiobissulfosuccinimidyl propionate (Pierce Chemical Co., Rockford, Ill., USA)for 30 minutes at 37° C., and lysed.

PTN-Fc “Capture”

U373-MG cells (≈10⁶) were lysed as above, and proteins associated withPTN-Fc were bound to Protein A Sepharose-4B and, after washing, elutedby boiling in sample buffer and analyzed in Western blots as above.

Glutathione S-Transferase (GST) “Capture” Assays

The GST-juxtamembrane (D1) fragment and the D1 fragment Cys-1925-Serwere prepared by using a human RPTP β/ζ cDNA fragment to encode aminoacids 1655–2018 fused with GST in the expression plasmid PGEX-KG XhoIand XbaI sites. The constructs (or GST alone) were expressed in BL-21competent cells from 5 ml overnight cultures, and the recombinantproteins were immobilized with 100 μl of glutathione-Sepharose-4B beads(Amersham Pharmacia). The beads were then incubated with U373-MG celllysates from 60-mm confluent dishes, washed, eluted, and analyzed inWestern blots as above.

Antibodies and Other Reagents

α-β-catenin and α-RPTP β/ζ antibodies were obtained from TransductionLaboratories (Lexington, Ky.), and α-phosphotyrosine monoclonalantibodies (4G10) were obtained from Upstate Biotechnology (Lake Placid,N.Y., USA). Recombinant PTN was purchased from SIGMA Chemical Company(St. Louis, Mo., USA), and recombinant PTN-Fc was purified fromconditioned media of human embryonic kidney 293 cells expressing a cDNAthat encodes the full-length PTN molecule fused at its C terminus withthe Fc fragment of IgG. For the dose responses of both PTN and PTN-Fc toinactivate RPTP β/ζ, see below. PTN was used at 50 ng/ml and PTN andPTN-Fc were established by using tyrosine phosphorylation of β-cateninand the ability PTN-Fc was used at 5 ng/ml, saturating levels of each,respectively.

Example 2 RPTP β/ζ Tyrosine Phosphatase Activity

PTN-treated U373-MG cells. Confluent U373-MG cells were incubated eitherwith DMEM alone or 50 ng/ml recombinant PTN (Sigma) at 37° C. for 15minutes, washed three times with PBS, lysed as described above, andcleared at 14,000×g for 15 minutes 4° C. Equal amounts of lysates wereincubated with α-RPTP β/ζ antibodies or mouse IgG (control) at 4° C.overnight, incubated with Protein A Sepharose-4B at 4° C. for 2 hours,and washed three times in lysis buffer and once in assay buffer (20 mMimidazole, pH 7.2/0.1 mg/ml BSA). The phosphatase activity of theimmobilized RPTP β/ζ protein was assayed as follows: 50 μl of eitherRPTP β/ζ or mouse IgG immobilized on protein-A beads was added to theassay buffer, the reaction mixture (25 mM imidazole, pH 7.2/0.1 mg/mlBSA/10 mM DTT/100 nM ³²P-labeled substitute Raytide) was added to afinal volume of 80 μl, incubated at 30° C. for various times,terminated, and the ³²P released was quantitated by a charcoal-bindingassay. The synthetic peptide Raytide (Oncogene Science, Inc., Uniondale,N.Y., USA) was phosphorylated at its unique tyrosine residue byfollowing the manufacturer's instructions.

RPTP β/ζ activity in Sf9 cell membranes. The Bac-to-Bac BaculovirusExpression System (Life Technologies, Gibco/BRL, Gaithersburg, Md., USA)was used to express RPTP β/ζ in Sf9 cells. A full-length human RPTP β/ζcDNA was cloned into a pFastBac donor plasmid at NotI and XbaI sites(pFastBac-RPTP β/ζ), transformed into DH10Bac Escherichia coli whichcontains bacmid and helper virus, and plasmid DNA prepared. Sf9 cellswere infected by the recombinant virus according to the manufacturer'sinstructions. To prepare membrane fractions, cells were sonicated in ahypotonic lysis buffer (25 mM Tris-HCl, pH 7.5/25 mM sucrose/0.1 mMEDTA/5 mM MgCl₂/5 mM DTT/1 mM phenylmethylsulfonyl fluoride/0.5 μg/mlleupeptin/1 μg/ml aprotinin), nuclei were removed by low-speedcentrifugation, and membrane fractions were obtained by centrifugationat 100,000×g for 60 minutes at 4° C. The resulting pellets weresuspended by sonication in lysis buffer, brought to a concentration of 2mg/ml, and used to measure PTPase activity as above.

The assays were linear with time and protein concentration.

Example 3 Both Exogenous PTN and the Endogenous Ptn Gene ProductInteract with RPTP β/ζ

PTN-Fc was incubated with lysates of serum starved, confluent U373-MGcells, and proteins associated with PTN-FC were captured on Protein ASepharose and probed by Western blot with anti-(α)-RPTP β/ζ antibodies(FIG. 1B). Three major and other minor alternative-spliced forms of thesingle RPTP β/ζ gene have been identified (28, 29, 30), and the resultsof the PTN-FC capture were therefore compared with Western blots ofimmunoprecipitates from untreated U373-MG cell lysates incubated withA-RPTP β/ζ antibodies (FIG. 1A). Major bands of ≈230, ≈130, ≈85, andvariably, in other experiments, ≈250 kDa were identified (FIG. 1A),consistent with the known different spliced forms of RPTP β/ζ previouslyidentified. Depending on the conditions of cell growth, different(presumably alternative-spliced) forms were identified. In Western blotsof proteins captured by PTN-Fc from U373-MG cell lysates, two majorbands of ≈130 and 85 kDA were identified (FIG. 1B), suggesting thatPTN-Fc preferentially associates with isoforms of ≈130 and ≈85 kDa.However, when the blots were exposed for longer times, a faint band at230 kDa was also seen. When IgG alone was substituted for PTN-Fc, RPTPβ/ζ was not captured by Protein A Sepharose (FIG. 1B, left lane). Whenblots were reprobed with α-IgG antibodies that recognize the Fc portionof PTN-Fc or anti-PTN antibodies, it was established that PTN-Fc waspresent in the complex captured by Protein A Sepharose.

PTN itself is also expressed in U373-MG cells. To show that theendogenously expressed PTN and RPTP β/ζ interact with each other invivo, untreated U373-MG cell lysates were immunoprecipitated with α-PTNantibodies and analyzed in Western blots. Anti-RPTP antibodiesrecognized protein bands at 130 and 85 kDa (faint) (FIG. 1C). Theseresults thus establish that both exogenous and endogenous PTN physicallyinteract with the major alternatively spliced products of RPTP β/ζ inU373-MG cells.

Example 4 PTN Inactivates RPTP β/ζ Activity in vivo and in vitro

To directly determine if PTN affects the function of endogenous RPTPβ/ζ, lysates of PTN-treated and control, untreated U373-MG glioblastomacells were immunoprecipitated with α-RPTP β/ζ antibodies, incubated withProtein A Sepharose, and directly assayed for protein tyrosinephosphatase activity as described above (FIG. 2A). The effects of PTN onthe catalytic activity of recombinant RPTP β/ζ also were tested by usingmembrane fractions prepared from Sf9 insect cells infected with abaculovirus expressing recombinant RPTP β/ζ. Remarkably, the proteintyrosine phosphatase activity of the endogenous RPTP β/ζ inimmunoprecipitates from PTN-treated cells was reduced by more than 90%when compared with RPTP β/ζ from untreated cells and when corrected fornon-specific background (IgG controls, FIG. 2A). PTN also strikinglyreduced the catalytic activity of recombinant RPTP β/ζ in Sf9 membraneswhen background phosphatase activity was again corrected (FIG. 2B). Theinhibition by PTN is specific, because PTN inhibits tyrosine phosphataseactivity only in Sf9 cell membranes that express RPTP β/ζ and theinhibition is rapid (FIG. 2C). Nearly 70% of the phosphatase activity orRPTP β/ζ is lost in 5 minutes. Thus, PTN not only physically associateswith RPTP β/ζ but functionally, PTN profoundly reduces the catalyticactivity of RPTP β/ζ. Furthermore, because PTN effectively reduces theendogenous RPTP β/ζ activity, it can be concluded that RPTP β/ζ is anintrinsically activity tyrosine phosphatase, thereby suggesting thatRPTP β/ζ may be an important regulator of steady-state tyrosinephosphorylation of compatible intracellular substrates that themselvesare regulated by an intrinsically active tyrosine kinase activity.

Example 5 β-Catenin is a Potential Substrate for RPTP β/ζ

β-catenin is known to associate with other RPTPs and be phosphorylatedin tyrosine (31, 32). β-catenin also is an important signaling moleculein development and in the wnt/APC−/− oncogenic pathways (33, 34, 35),suggesting that its signaling properties may be influenced by tyrosinephosphorylation and potentially be regulated by RPTP β/ζ and/or PTN.PTN-Fc-treated U373-MG cells were therefore incubated with3,3′-dithiobis-sulfosuccinimidyl-propionate and lysed. Proteinscross-linked to PTN-FC were captured with Protein A Sepharose andanalyzed by Western blot with either α-β-catenin (FIG. 3A, right) orα-RPTP β/ζ antibodies (control, FIG. 3A, left). In SDS/PAGE gels, ahigher molecular weight complex with very limited migration wasidentified, and immunoreactive PTN and RPTP β/ζ were identified in thisband. In other control experiments, both β-catenin (FIG. 2A Right, lane3) and RPTP β/ζ (FIG. 3A Left, lane 3) were readily recognized inuntreated U373-MG cell lysates. When the captured protein complex fromPTN-Fc treated cells cross-linked with3,3′-dithiobis-sulfosuccinimidyl-propionate was reduced before SDS/PAGEand analyzed in Western blots probed with α-RPTP β/ζ antibodies, RPTPβ/ζ-spliced forms of ≈130 kDa, and more weakly, ≈230 kDa, wereidentified (FIG. 3A, Left, lane 1). These forms were not identified inlysates of cells treated with the Fc fragment of IgG alone (FIG. 3Aleft, lane 2). Remarkably, PTN-Fc also captured β-catenin, based onrecognition by α-β-catenin antibodies and the migration of the bandrecognized by α-β-catenin antibodies and the migration of the bandrecognized by α-β-catenin at the estimated molecular mass of β-catenin(≈94 kDa) (FIG. 3A Right lane 1). β-catenin was not captured when cellswere treated with the Fc fragment of IgG alone (FIG. 3A Right, lane 2).The results confirm that the extracellular domain of RPTP β/ζ interactswith PTN-Fc and suggest that β-catenin interacts with its intracellulardomain. These results also raise the possibility that RPTP β/ζ links PTNsignaling to β-catenin.

RPTP β/ζ has two phosphatase domains in its C-terminal cytoplasmic tail.The juxtamembrane-proximal D1 domain of RPTP β/ζ contains an activetyrosine phosphatase catalytic unit whereas the juxtamembrane-distal D2domain lacks the required cysteine residue and thus is inactive (36). Tosee whether β-catenin associates with the active site of RPTP β/ζ, theD1 domain and the D1 domain Cys-1925-Ser (active site inactivating)mutation were coupled with GST, incubated for 15 minutes with U373-MGcell lysates from cells pretreated with pervanidate, and analyzed inWestern blots. Both the active and inactive D1 domains of RPTP β/ζβ-catenin (FIG. 3B, upper) at essentially equal levels. However, whenthe Western blots were reprobed with α-phosphotyrosine antibodies, thelevels of tyrosine phosphorylation of β-catenin were sharply reduced inlysates incubated with the active (wt) D1 domain compared with the D1domain Cys-1925-Ser (FIG. 3B, lower), localizing the association ofβ-catenin to the active site-containing D1 domain and stronglysuggesting that β-catenin is a substrate of RPTP β/ζ.

Example 6 PTN Stimulates Tyrosine Phosphorylation of β-Catenin

To pursue the possibility that β-catenin is a substrate of RPTP β/ζ inintact cells and that PTN-dependent inactivation of RPTP β/ζ influencestyrosine phosphorylation of β-catenin, tyrosine phosphorylation wasexamined temporally after the addition of PTN-Fc to intact U373-MGcells. Tyrosine phosphorylation of β-catenin increased within 2 minutesof addition of PTN and reached peak levels within 8 minutes (FIG. 4A).The levels of β-catenin itself were essentially identical, indicatingthat PTN-Fc had no detectable influence on the levels of β-cateninprotein. Furthermore, the response was PTN-Fc dose-dependent between 0.2and 5 ng/ml (FIG. 4B).

All references, patents and patent applications are incorporated hereinby reference in their entirety. While this invention has beenparticularly shown and described with references to preferredembodiments thereof, it will be understood by those skilled in the artthat various changes in form and details may be made therein withoutdeparting from the spirit and scope of the invention as defined by theappended claims.

1. A method of reducing levels of tyrosine phosphatase activity ofprotein tyrosine phosphataseζ/receptor-like protein tyrosine phosphataseβ (RPTP β/ζ) in a mammalian cell in vitro that expresses RPTP β/ζ, themethod comprising administering to the cell in vitro a mammalianpleiotrophin of about 18 kDa in an amount effective to reduce thetyrosine phosphatase activity of the RPTP β/ζ.
 2. A method of increasingtyrosine phosphorylation of β-catenin in a mammalian cell that expressesRPTP β/ζ, said method comprising contacting the cell that expresses theRPTP β/ζ with a mammalian pleiotrophin of about 18 kDa in vitro in anamount effective for reducing tyrosine phosphatase activity of the RPTPβ/ζ, thereby increasing tyrosine phosphorylation of the β-catenin. 3.The method of claim 3, wherein the amount of the mammalian pleiotrophineffective for reducing tyrosine phosphatase activity of the RPTP β/ζ isan amount of the pleiotrophin effective for inducing ligand-dependentdimerization of the RPTP β/ζ.
 4. The method of claim 3 wherein theamount of the mammalian pleiotrophin effective for inducingligand-dependent dimerization of the RPTP β/ζ is an amount of thepleiotrophin effective for inhibiting the ability of the β-catenin tobind to the catalytic site of the RPTP β/ζ.
 5. The method of claim 4wherein the catalytic site of the RPTP β/ζ is the D1 domain of the RPTPβ/ζ.
 6. The method of claim 1, wherein the mammalian pleiotrophin isselected from the group consisting of a bovine pleiotrophin, a humanpleiotrophin, a rat pleiotrophin and a mouse pleiotrophin.
 7. The methodof claim 1, wherein the mammalian pleiotrophin is a human pleiotrophin.